The mammalian retina is composed of a diverse mixture of cell types with distinct functional roles (e.g. photoreceptors and multiple types of interneurons). The properties of distinct retinal cell types appear to be specified by different underlying patterns of gene expression, but the mechanisms that establish and maintain specific patterns of gene expression in individual retinal cell types are only partially understood. The recent discovery of an abundant class of small RNA regulatory molecules in animals, microRNAs (miRNAs), provides a potential novel regulatory mechanism for retinal cell diversification. Although several hundred miRNAs have been found to be expressed in the mammalian nervous system, recent computational searches for genes that encode miRNAs, as well as sequencing of miRNAs expressed in mammalian brain, suggest that numerous mammalian miRNAs remain to be identified. Genetic studies in the nematode C. elegans have identified miRNAs that control the identity of specific neurons and some of these miRNAs are expressed in very small populations of cells. If similar miRNAs, restricted to specific cell types, exist in the mammalian retina, those miRNAs from less abundant cell types will represent a small fraction of all miRNAs and therefore are unlikely to have been identified to date. The specific goals of the proposed project are to create an extensive profile of miRNAs expressed in the adult and neonatal mouse retina, to identify miRNAs that are restricted to specific retinal cell types or subtypes, and to identify retinal miRNAs with altered expression in the retinal degeneration 1 (Pde6brd1) mouse mutant, a model of human retinitis pigmentosa. miRNAs will be identified by extensive or deep sequencing, using a massively parallel sequencing technology. Several million short RNAs from adult and neonatal wild-type retinas and from adult Pde6brd1 retinas will be sequenced (>1000-fold deeper sequencing than prior analyses of retinal miRNAs). Sequences will be computationally analyzed to identify known and novel miRNAs and to exclude other RNAs. The miRNAs identified in the retina will be further characterized using recently developed miRNA in situ hybridization techniques. Based upon the frequency of different cell types in the retina, the deep sequencing of retinal miRNAs should allow the identification of miRNAs restricted to low abundance retinal cell types, as well as the generation of a comprehensive profile of miRNAs expressed in the retina. The proposed studies are intended to provide a framework for understanding miRNA expression and function in the mammalian retina. Identification of cell-type specific and other miRNAs in the retina will provide new genes that can be considered as candidates for human retinal diseases. In addition, expression of cell-type specific miRNAs may be subject to modulation in retinal diseases.